Aug ., 1936
COMMUNICATIONS TO THE EDITOR
uncorrected but were taken in a Fisher melting point apparatus with a set of Anschiitz thermometers which gave correct melting points with
1503
various pure reagents. DEPARTMENT OB CHEMISTRY HOLYCROSSCOLLEGE
RECEIVED JUNE1, 1936
woRCBSTER, M~~~~~~~~~
COMMUNICATIONS T O T H E EDITOR SYNTHETIC SUBSTRATES FOR PROTEIN-DIGESTING ENZYMES
TABLEI ENZYMIC HYDROLYSIS OF WHIPPURYL-LYSINE-AMIDE
Sir: Knowledge regarding the specificity of those enzymes which hydrolyze intact proteins (peptic, tryptic and catheptic proteinases) is meager. In general it is assumed that these enzymes react exclusively on high molecular substrates. Recently it has been possible to study the specificity of proteinases with the aid of synthetic substrates. Such substrates have been found in this Laboratory for the catheptic enzymes, papain, liver-cathepsin and bromelin. The authors have now observed the splitting of a-hippuryl-lysineamide by tryptic proteinase. a-Hippuryl-e-carbobenzoxy-lysine methyl ester was converted into cu-hippuryl-ecarbobenzoxylysine amide, m. p. 212O, with the aid of methanolic ammonia. AmE. Calcd. for &HzsN405: C, 62.7; H, 6.4; N, 12.7. Found: C, 62.6; H, 6.7; N, 12.8. This amide was hydrogenated catalytically, yielding a-hippuryl-lysine-amide which was isolated as the strongly hygroscopic hydrochloride, m. p. 248’. Anal. Calcd. for C1bH23N*O&l: C, 52.5; H, 6.8; N, 16.3. Found: C, 52.0; H, 7.0; N, 15.9. The tryptic proteinase was prepared according to E. Waldschmidt-Leitz and A. Purr [Ber., 62, 2217 (1929)l. The preparation contained no dipeptidase, aminopeptidase, and no carboxypeptidase ; however, protaminase probably was present (Table I). In contrast to HCN-papain, which splits only one peptide bond, tryptic proteinase splits two. After a complete splitting, hippuric acid was isolated from the digest (over 70% of the theoretical amount). Therefore, the splitting also must have liberated lysine and ammonia. That the free eamino group is an essential condition for the enzymic hydrolysis is shown by the fact that the
AT
(Titration of
40’
liberated carboxyl groups)
Enzyme
Tryptic proteinase, PH 8.8
Time, hrs.
22 72
Tryptic proteinase, PH 8.8
18
HCN-Papain, pH 5.0
42 5 24 49
Hydrolysis in % of one peptide bond
123 200 121 175 58 80 85
above mentioned a-hippuryl-e-carbobenzoxy-lysine amide is not hydrolyzed under the conditions of our experiments. The hydrolysis of our substrate by tryptic proteinase is remarkable since tryptic proteinase is supposed to react exclusively on anionic substrates. I t is intended to continue this research by studying the action of pure tryptic proteinases. TF~ELABORATORIES OF M A XBERGWN THE ROCKEFELLER INSTITUTE WILLIAM F. Ross FOR MEDICAL RESEARCH NEWYORK,N. Y. RECEIVED JULY10, 1936
STEROLS. VI. SYNTHETIC PREPARATION OF OESTRONE (THEELIN)
Sir: The evidence for the accepted structure of oestrone has recently been reviewed [L. F. Fieser, “Chemistry of Natural Products Related to Phenanthrene,” A. C. S. Monograph Series, No. 701. We have been able to prepare a well crystallized compound from ergosterol which by analysis, derivatives and mixed melting points, is identical with oestrone isolated from pregnancy urine. It has been previously shown that ergosterol may be converted into 3-hydroxy-nor-allo-cholanic acid [Chuang, Ann., 500, 270 (1933); Fernholz
